Method for recharging an ion exchange resin

ABSTRACT

A method for recharging and purifying an acetate group ion exchange resin which has been used to isolate thyroxine from blood serum. The method includes treatment with an aqueous concentrated acetic acid solution optionally followed by treatment with an aqueous metallic acetate solution.

United States Patent 51 Apr. 25, 1972" Young [54] METHOD FOR RECHARGINGAN ION EXCHANGE RESIN [72] Inventor: David P. Young, South SanFrancisco,

Calif.

[73] Assignee: Technicrait International, Inc., San Matio,

Calif.

[22] Filed: Mar. 16, 1970 [2]] Appl.No.: 20,135

[52] use: ..2-1o/32,210/37 [51] Int.Cl v v ..B0 ld,l5/06 [58]FieIdoiSearch ..2lO/32,37;260 /5l9 Reierences Cited UNITED STATESPATENTS 3,471,553 l0/l969 Bittner ..260/5l9 [57] ABSTRACT A method forrecharging and purifying an acetate group ion exchange resin which hasbeen used to isolate thyroxine from blood serum. The method includestreatment with an aqueous concentrated acetic acid solution optionallyfollowed by treatment with an aqueous metallic acetate solution.

4 Claims, No Drawings METHOD FOR RECHARGING AN ION EXCHANGE RESINBACKGROUND OF THE INVENTION Thyroxine, an organic iodide, is theprincipal active hormone component secreted by the thyroid gland. It iscirculated by blood serum through the body. A quantitive determinationof thyroxine, based on its iodine content, may be accomplished byisolation from serum contaminants (i.e., all iodine-containing organicand inorganic substances other then thyroxine) followed by analysis ofthe iodine content of the thus-isolated thyroxine. Presently, suchisolation is being performed extensively in the laboratory bychromatography on ion exchange resin columns (commonly designated T-4 bycolumns) by sequential elution with varying concentrations of aceticacid. The inorganic iodide contaminants of serum are firmly retained onthe column at any acid concentration. The organic contaminants are firsteluted with a relatively dilute acid wash and discarded. Finally,thyroxine is eluted with a more concentrated acid wash.

The aforementioned method for isolation and quantitation of thyroxine isdescribed as an automated integral procedure in Bittner, D., et al., AnAutomated Method for the Bromination and Quantitation of Thyroxine inResin Column Eluates," published for the Thyroxine Round Table, 1968Annual Meeting, American Society of Clinical Pathologists (Oct. 14, 1968at Miami, Florida). According to the Bittner technique, isolation isperformed using a resin of the trademark type AGl,X, 100-150 mesh resinproduced by BioRad Laboratories, Richmond, California. This stronglybasic anion exchange resin is composed of quaternary ammonium exchangegroups attached to a styrenedivinylbenzene polymer lattice. Although theAG-l resin is originally produced in the chloride form, it is convertedto the acetate form for purposes of thyroxine separation. The isolationtechnique described in the Bittner article is performed on this AG-lresin in acetate form and includes the following general steps: dilutingserum with alkaline medium to dissociate thyroxine from serum protein;depositing the diluted'serum into a column of the aforementioned resin;and washing the column with a series of acetic acid solutions of varyingpH values. In the first washes, protein, monoiodotyrosine,diiodotyrosine, and some organic contaminants are removed whilethyroxine, thyronine, and inorganic iodine remain on the resin. In asecond dilution, at a higher concentration of acetic acid, the thyroxineis eluted and quantitated. Although Bittner, et al. described anautomated method, thyroxine isolation and quantitation frequently isperformed manually. Isolation on the T-4 by column is fast, efficientand accurate.

One drawback of using such columns is that presently there is noavailable method for reusing the columns after one complete T-4 elutioncycle. In fact, all commercial manufacturers of the column specificallyinstruct purchasers not to reuse the column. One explanation given forthis instruction is quite simple; there is a good chance that organiciodide contaminants which were not eluted in the isolation of the firstserum sample could be eluted during isolation of the second thyroxinesample. As already noted, these contaminants(or any iodine-containingsubstance) would interfere with thyroxine quantitation which is based onthe iodide content of the column eluate. Another explanation is thatsuch organic iodide contaminants deplete the resin acetate ion groupsavailable for ion exchange during elution of the second sample.

SUMMARY OF THE lNVENTlON AND OBJECTS This invention relates to a methodfor recharging and purifying an acetate ion-exchange resin partiallydepleted and contaminated by the presence of organic iodide retainedthereon.

According to the invention, contaminated resin particles (e.g.,following a thyroxine isolation therewith, as described hereinafter, aresubjected to treatment with highly concentrated acetic acid for thepurpose of recharging and purifying the resin for repeated use. Thistreatment requires immersion of the resin particles in an aqueous aceticacid solution at an acid concentration substantially greater than 50%and preferably greater than 70%. If the resin is particularlycontaminated, the thus-treated resin particles may be drained andretreated with a metallic acetate solution at a molar concentration ofat least 0.5 M in sufficient quantity for essentially complete resinimmersion. Both treatments are performed to disengage the organic iodidecontaminants ionically held by the resin followed by elution thereof andto recharge the thusvacated ionic groups of the resin with acetate ions.

It is an object of the present invention to provide a rapid andeconomical method for recharging and purifying an acetate group ionexchange resin which has been partially depleted and contaminated by thepresence of organic iodide.

in particular, it is an object of the present invention to .provide amethod of the above type which enables a used T-4 by column to beemployed in the isolation of thyroxine from a subsequent serum sample.

Additional objects and features of the invention will appear from thefollowing description.

DETAlLED DESCRIPTION OF THE PREFERRED EMBODIMENT In the presentinvention a particulate acetate ion exchange resin which has been usedin a T-4 by column for the isolation of thyroxine from protein isrecharged for repeated thyroxine isolation therewith. In a primaryrecharge treatment the resin is immersed in a solution containing a highconcentration of acetate ions. For this purpose, the treating solutionis preferably aqueous acetic acid at a concentration substantiallygreater than 50% by volume, more particularly greater than 70%. Aftersuch primary treatment the resin may be drained and treated with asecondary acetate ion from a source such as a metallic acetate solutionwith a molar concentration of at least 0.5 M.

The method of the present invention will be described in terms ofrecharging an acetate ion exchange resin of the aforementioned AG-ltype. However, it is to be understood that any ion-exchange resin whichis in acetate form .may be employed. For example variation of polymerlattice attached grouping thereon, and resin mesh size are deemed to bewithin the scope of this invention.

The primary recharge solution, acetic acid, may vary in concentrationfrom an aqueous solution of 50% acid up to acid (glacial acetic acid).The concentrations of the acid treat ment is usually somewhat higherthan-the concentration of the recharge solution because after beingsubmerged in liquid for prior isolation the resin particles are normallyincompletely drained under the force of gravity. Thus the rechargesolution is diluted by the liquid retained in the resin yielding asomewhat lower overall acid concentration for treatment. For example, ifglacialacetic acid were added to the resin in a ratio of one part byvolume acid to one part by volume of retained distilled water, thetreating acid, as used herein, would be at 50% concentration.

The function of the optional secondary treatment with a metallic acetatesalt is to provide an additional source of acetate ion as a safeguard,if necessary, to assure that the resin is purified and recharged.Although this safeguard normally is not required, itwould be most usefulwhere the blood serum is known to contain a high degree ofcontamination. Any highly ionized metallic acetate aqueous solution maybe used such as a solution containing the acetate salts of sodium,potasium, calcium, magnesium, zinc or combinations thereof. For optimumeffectiveness, the secondary treating solution should have aconcentration of at least 0.5 M solutions containing as high as 4.0 Msodium acetate are particularly effective.

The acetate resin to be recharged must be in contact with the rechargesolution for a sufficient period of time for the acetate ions of therecharge solution to replace contaminants in the resin. It has beenfound that the time for elution under the force of gravity, on the orderof 30 seconds, is sufficient contact time. However, it is preferred toplace the resin in a recharge solution for a substantial period of time(e.g., 12 hours) to make certain that all the contaminants replaceableby the acetate ions are driven off the resin and withdrawn with thewashing effluent for discard prior to performance of the second run.

The amount of recharge solution to be used depends upon the acetate ionconcentration thereof. Although an amount on the order of 3.5 ml ofconcentrated acetic acid would normally contain sufficient acetate ionto recharge and purify the resin, it is preferred to employ at least 7ml of concentrated acetic acid for recharging, especially if the priorthyroxine isolation were performed on an abnormal blood sample asdefined hereinafter.

In order to more clearly disclose the nature of the present invention, aspecific detailed description of a first separation by means of a T-4 bycolumn is herein given. it should be understood, however, that this isdone by wayof example and that modifications of procedure'and of the T-4column itself may be employed in the isolation procedure prior toperformance of the present invention.

in one embodiment, thyroxine is separated on a T-4 by column formed of acylindrical resin with an internal diameter of 8.0 mm and a height of2.5 cm. An anion-exchange resin formed of A6 1, X2, 100-150 mesh, withat least 90% of the quaternary ammonia groups converted to the acetateform. The total amount of added resin is on the order of l'l.5

grams.

The exchange resin is first made alkaline by washing with sodiumhydroxide reagent (e.'g., formed by dissolving8 grams of sodiumhydroxide in 2 liters of water). Blood serum (0.5 ml) is diluted withapproximately 6 ml of sodium hydroxide reagent of the same type andquantitatively passed through the aforementioned ion exchange column.Prior studies have demonstrated that essentiallyall of the serumthyroxine is effectively isolated on the column at this time. v

The column is then washed with 2 aliquots (approximately 7 ml each) ofacetate alcohol wash solution. This solution may be prepared bydissolving 35 grams of sodium acetate in 1,500 ml of water, and adding400 ml of methyl alcohol, adjusting the pH to 5.5 with concentratedacetic acid, and adding water to the 2 liter volumetric mark. Allefiluents of this wash solution are discarded. in a second washingstage, the column is washed with 2 aliquots (about 7 ml each) of.15%,acetic acid.

All effluents discarded.

During the aforementioned first and second washing stages, proteins,monoiodotyrosine, diiodotyrosine and certain other organic contaminantsare removed while thyroxine, traces of thyronine, and inorganic iodidesremain on the resin.

The column is then primed for thyroxine solution by the addition of 0.6ml of concentrated acetic acid thereto. During this step, the remainingtraces of thyronine and other contaminants are essentially eliminated inthe effluent while the thyroxine is moved from the top to the bottomportion of the resin without elution therefrom. This effluent isdiscarded and the column allowed to drain as completely as possibleunder the force of gravity. Even thyroxine, the strongest held of thecommonly occurring iodide compounds in blood serum, is moved downwardlyunder the action of this higher acetate concentration.

To elute the thyroxine, about 3 ml of 50% acetic acid is added twice tothe column to form a first and second effluent. The only source ofiodine in these effluents should be thyroxine. An iodine analysis maythen be performed on each eluent to determine the original thyroxineconcentration. It is noted that errors in this determination would beintroduced by the presence of any other iodine-containing compound. Onesuch analytical technique for quantitation of thyroxine may utilize theautomated method described in the aforementioned Bittner etallpublication.

exchange resin and is not eluted by the above described isola- 7 tionmethod. Therefore, such iodide does not interfere with quantitation ofthyroxine. However, it is reasonable to assume that certain organiciodide contaminants in blood serum are also retained on the column andnot eluted during the abovedescribed elution process. if it wereattempted to use the by column for a second thyroxine quantitation, suchretained organic iodides could be eluted along with the thyroxine toproduce substantial error in quantitation. This is particularly truewith so-called abnormal blood samples, containing greater than 20microgram percent (i.e., micrograms per ml of whole blood). In fact, ithas been found that a first isolation of an abnormal blood sample doesleave contaminants on the column which produce errors when a secondblood sample were run through the column for purposes of thyroxine quan-11181101'1.

According to the invention, the above column which is considered by theT-4 by column industry to be contaminated and of no use, may berecharged for use on the order of 3 to 7 times depending on theeffectiveness of the recharge solution.

In one method of the invention, a column, contaminated as above, isfilled with distilled water and allowed to drain completely. This stepis performed to wash out any water soluble impurities that might beretained on the sides of the column or in the resin after thyroxineisolation of the prior sample.

During the recharging step, the column may be washed with a rechargesolution of approximately 7 ml of concentrated acetic acid. To assurecomplete mixing, the resin and recharge solution should be agitated asby stirring. Following agitation,

the recharge solution is retained on the column for a period of timesuch as 10 minutes. Then, the solution is drained and discarded tocomplete the recharge. Thereafter, the column may be used forquantitation of a second blood serum sample for thyroxine.

In order to determine the effectiveness of the recharge method of thepresent invention, 20 different normal and abnormal serum specimens wereutilized. Each specimen was diluted with a solution of low-ionic contentcontaining a trace of I-l3l labeled thyroxine for elution control anddivided into three equal portions (0.5 ml of serum per tube) labeledsamples A, B, and C." Then thyroxine from the A" samples was isolated,each on a separate T4 column and quantitated in a manner as describedabove. Each of the used A" columns were then recharged and purified asabove described. The B samples specimens were then isolated on thecorresponding recharged columns and quantitation of the thyroxinetherein was quantitated. To provide an independent comparison, thethyroxine in the C" samples was then isolated on a new column andquantitated. The quantitative values obtained for each of the A, B, andC samples are tabulated in the table below. The numerical values listedrepresent microgram percent of thyroxine.

Sample A Sample B Sample C Specimens Micqzogram Microgram MicrogramNormal specimens 3.7 3.5 3?; 6.5 6.7 6.5 7.4 7.2 7.0 5.6 5.8 5.5 6.1 6.16.

Abnormal specimens 2 5'9 (low thyroxine 1.3 1.2 1.2 value) 2.0 1.8 2.02.4 2.5 2.7 1.6 1.7 1.5 1.9 2.0 2.0 2.0 2.4 Abnormal specimens 1.1 09(high thyroxine 9.8 10.0 10.1 value) 8.5 8.9 8.7 10.5 10.3 10.5 11.511.4 11.7 12.0 12.3 12.1 13.9 14.1 13.9

It can be seen from the above table that the thyroxine values weresubstantially the same upon quantitation for each of the samples A, B,and C. Thus, the recharge column, used for sample B, performs withessentially the same degree of efficiency as does a fresh column.Consequently by use of the recharge technique of the present invention,one may reuse a column without contamination from a prior isolation onthe same column.

it is apparent from the foregoing that a method has been provided forrecharging a used T-4 by column. It is obviously more economical toutilize the inexpensive recharge solution to obtain two or morethyroxine isolations on a single T-4 column than to discard therelatively expensive column after one use.

lclaim:

l. A method for the sequential isolation of thyroxine from at least twoblood serum samples by selective adsorption on a single column of anacetate group anion exchange resin comprising the sequential steps of:

a. isolating thyroxine from'a first serum sample on a column of theanion exchange resin;

b. eluting a first thyroxine fraction from said column with a volume ofacetic acid;

c. eluting a second fraction of thyroxine from said column with asufficient second volume of acetic acid so that small quantities ofiodine source contaminants remain on said resin;

d. treating said column with a sufficient quantity of acetic acidrecharge solution to obtain essentially complete resin immersion, saidrecharge solution having an acetic acid content substantially greaterthan 50% by volume;

e. maintaining contact between said resin column and said rechargesolution for a sufficient period of time for the acetate ions of therecharge solution to replace contaminants in the resin;

f. draining said recharge solution from said column;

g. isolating thyroxine from a second serum sample on the same column ofanion exchange resin; 1

h. eluting thyroxine from said column.

2. A method as in claim 1 wherein said acetate groups are ionicallybonded to quaternary ammonia groups forming a portion of said resin.

3. A method as in claim 1 in which the acid concentration of solutionadded in step (d) is at least 70% by volume.

4. A method as in claim 1 including the additional steps of retreatingsaid drained resin particles from step (f) with an aqueous mixturecontaining a metallic acetate solution at a molar concentration of atleast 0.5 M in sufficient quantity for essentially complete resinimmersion, and draining said aqueous mixture from said column.

2. A method as in claim 1 wherein said acetate groups are ionicallybonded to quaternary ammonia groups forming a portion of said resin. 3.A method as in claim 1 in which the acid concentration of solution addedin step (d) is at least 70% by volume.
 4. A method as in claim 1including the additional steps of retreating said drained resinparticles from step (f) wIth an aqueous mixture containing a metallicacetate solution at a molar concentration of at least 0.5 M insufficient quantity for essentially complete resin immersion, anddraining said aqueous mixture from said column.